The kinetics of both intracellular and extracellular beta-lactamase production and the relationsh... more The kinetics of both intracellular and extracellular beta-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacterium fortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in beta-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of beta-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. Howeve...
Twenty-five strains of Mycobacterium avium-intracellulare (MAC) isolated from acquired immunodefi... more Twenty-five strains of Mycobacterium avium-intracellulare (MAC) isolated from acquired immunodeficiency syndrome (AIDS) patients in three medical centres in Italy have been studied. Serotyping performed on eighteen strains showed various serovars within either M. avium or M. intracellulare serotypes and with serovars 1 and 21 being the most prevalent (four strains for each serovar). Among fourteen drugs used for testing the antibiotic sensitivity, rifapentine, rifabutin and clofazimine showed to have the best in vitro activity. In an ex vivo model of infection using peritoneal resting macrophages from the C57BL/6 mouse, the intracellular viability of a strain of M. avium (strain 489, serovar 3) was reduced by clofazimine, amikacin, ciprofloxacin, rifabutin and clarithromycin (99, 98, 93, 89 and 69%, respectively), thus indicating for clofazimine a good correlation between in vitro and ex vivo activity.
Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs sh... more Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs showed nil to modest therapeutic activity in beige mice infected with Mycobacterium avium. However, the combination of all three, isoniazid-amikacin-clarithromycin, markedly reduced CFUs in both spleens and lungs after 91 days of infection.
The activity of clarithromycin and five other antimicrobial agents, namely amikacin, rifampicin, ... more The activity of clarithromycin and five other antimicrobial agents, namely amikacin, rifampicin, rifabutin, clofazimine and ciprofloxacin, was assessed both by an agar dilution and a radiometric method in broth on 11 Mycobacterium avium-intracellulare complex (MAC) strains, recently isolated from AIDS patients. Minimum inhibitory concentrations (MICs) radiometrically determined were, in general, several times lower than MICs assessed in agar, probably because of a partial degradation of antimicrobials during the long incubation period needed for tests in solid medium. When tested in broth, rifabutin and clofazimine showed very low MICs 90 (0.24 and 0.78 microgram/ml, respectively). Ciprofloxacin and clarithromycin also had MICs90 in the range of peak serum levels (1.93 and 3.76 micrograms/ml, respectively). Moreover, all these antimicrobials are known to concentrate several times in macrophages. MICs90 were higher for amikacin (11 micrograms/ml) and for rifampicin (8 micrograms/ml). When clarithromycin was tested against three MAC strains in combination with another drug, it showed a synergistic effect only when combined with rifampicin. Some synergistic effect was observed also when combining clarithromycin with rifampicin and amikacin, whereas in combination with rifabutin and clofazimine there was only an additive effect.
A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical propert... more A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called ‘beta-lactamase-stable’ cephalosporins.
Introduzione. La borreliosi di Lyme è un'infezione multisistemica trasmessa dalla zecca (Ixodes) ... more Introduzione. La borreliosi di Lyme è un'infezione multisistemica trasmessa dalla zecca (Ixodes) e causata dalla spirocheta Borrelia burgdorferi sensu latu (BB) che provoca patologie cutanee, e, se non trattata, cardiache, reumatologiche e neurologiche. Questa infezione è endemica nell'area del triveneto. La neuroborreliosi di Lyme (LN) è molto rara e la sintomatologia può a volte essere confusa con la malattia di Parkinson (PD) o altre patologie neurodegenerative. Metodi. Da un paziente (anni 57 M) con diagnosi clinica di PD deceduto per polmonite sono stati effettuati prelievi autoptici da corteccia cerebrale, cervelletto, ponte, nervo ottico, poligono di Willis, arteria coronaria, aorta toracica e addominale e arteria renale. I prelievi sono stati divisi in 2 parti, per l'estrazione e amplificazione del DNA (nested-PCR) e per PCR in situ. È stata ricercata una sequenza conservata del gene codificante per la flagellina di BB. Come controllo è stato utilizzato un ceppo di BB ATCC. Gli amplificati sono stati sequenziati per conferma. Risultati. Tutti i campioni, ad esclusione del poligono di Willis, sono risultati positivi all'amplificazione sia in provetta che in situ. La sequenza ha confermato la provenienza del DNA da BB. Conclusioni. Il paziente ha evidenziato la presenza di una infezione sistemica da BB e quindi potrebbe presumibilmente avere sviluppato una LN con sintomatologia analoga a quella associata al PD. Da questo studio emerge l'utilità di una valutazione sierologica e molecolare per la determinazione della presenza di BB in caso di sospetto di patologia neurodegenerativa non altrimenti spiegabile.
The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and... more The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography. The GroES preparation showed high purity by electrophoresis and immunoblotting. Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system. BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M. avium. Neither GroES nor CpG alone provided any protection against subsequent challenge with M. avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M. avium after intranasal challenge with a low dose of the organism. This indicates that intranasal administration of GroES and CpG o...
The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection rema... more The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the V␣14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of V␣14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1 ؉ V␣14 NKT population preferentially producing IFN-␥ predominated at an early stage (day 8), which was substituted by an NK1.1 ؊ population preferentially producing IL-4 at later stages (day 30). Despite the fact that V␣14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in V␣14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that V␣14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-␣ in liver and lungs of V␣14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-␣ mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for V␣14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.
The therapeutic effect of 18 anti-Mycobacterium avium regimens was examined in beige mice after 9... more The therapeutic effect of 18 anti-Mycobacterium avium regimens was examined in beige mice after 91 days of infection. Treatments included monotherapy with clarithromycin (CLA), ethambutol (EMB), amikacin (AMI), rifabutin (RFB), ciprofloxacin (CIP), clofazimine (CLO), and combinations of CLA, CLA-EMB, or CLA-AMI with one of the other drugs. After monotherapy, only AMI and CLA displayed bacteriostatic and/or moderate bactericidal effects in spleens and lungs, while CIP and RFB were totally inactive and CLO and EMB showed intermediate effects against the isolate tested. Resistant mutants were isolated in spleens of mice treated with EMB, CIP, RFB, and CLO. Among two-drug combinations, CLA-RFB, CLA-CIP, and CLA-CLO were significantly more active than RFB, CIP, CLO, respectively, but not more active than CLA alone, in both organs; CLA-AMI and CLA-EMB were bactericidal in spleens and lungs, respectively. Although activity of CLA-EMB was significantly potentiated by RFB and CLO in spleens and lungs, that of CLA-AMI was significantly increased by RFB and CLO only in lungs. The most active regimen in spleens and lungs on day 91 was the combination of all three, namely CLA-AMI-EMB, which reduced the CFU numbers of 2.7 and 7.5 logio; ui comparison with day 1 and day 91 counts in untreated control mice, respectively. an antimicrobial agent known to be bactericidal in vitro and in vivo against MAC911 showed activity similar to that of CLA when tested in a beige mouse model5 of disseminated infec-tion11 and prevented the emergence of resistant mutants.11,13 In the present study, we evaluated the anti-MAC activity of 18 different regimens, including monotherapy with CLA, AMI, EMB, rifabutin (RFB), ciprofloxacin (CIP), and clofazimine (CLO) and treatment with various two-and three-drug combinations, in the beige mouse model. In particular, we compared the efficacy of three-drug combinations obtained by combining CLA-AMI and CLA-EMB with one of the remaining drugs. Finally, the emergence of drug-resistant mutants during therapy with single or combinations of drugs was investigated.
BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Myc... more BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Me) and natural killer (NK) cell numbers increased 24.2 times and T-and B-cell numbers increased 31.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a 331.7-fold increase in Me and NK cells and a 39.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a 356.7-fold increase in Me and NK cells and a 222.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Me and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.
Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 ... more Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.
Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy... more Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4 + T cells producing IFN-g and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4 + T cell proliferation and IFN-g production in response to Ag85B protein, despite restraint of Th1 response by CD8 + T cell-dependent mechanisms and activation of CD4 + T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4 + T cells lost the ability to produce IFN-g and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4 + T cells determined dilution or loss of the protective Ag85B-specific CD4 + induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective antituberculosis immune response, and actually concur to disease progression.
It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhib... more It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhibited by mycobacteria results from the combination of factors including permeability to the drugs, beta-lactamase production, and affinity for penicillin-binding proteins (PBPs). We conducted an evaluation of the second and third factors by isolating nitrosoguanidine-induced mutants from the beta-lactamase-producing strain Mycobacterium fortuitum ATCC 19542 that displayed either elevated or reduced resistance to various beta-lactam antibiotics. The mutants studied included D1 (a beta-lactamase producer with high penicillin resistance), gamma 27 (a low-level beta-lactamase producer with low penicillin resistance), and D316 (a high-level beta-lactamase producer with high penicillin resistance). In all strains examined, four major PBPs, named 1, 2a, 2b, and 3, with apparent molecular weights of 102,000, 90,000, 87,000, and 50,000, respectively, were found. The MICs of various beta-lactams to...
Both smooth transparent (SmT) and smooth domed-opaque (SmD) colonial variants were obtained from ... more Both smooth transparent (SmT) and smooth domed-opaque (SmD) colonial variants were obtained from a strain of Mycobacterium avium isolated from a patient with AIDS. The two variants showed similar biochemical characteristics but SmT bacteria proliferated better than SmD bacteria inside human macrophages and were much less capable than the SmD variant of inducing the release of IL-lp, IL-6, TNF-a, GM-CSF and G-CSF, after incubation for either 3 or 6 days. As cytokines are important extracellular signals for immune cells, the lack of induction observed in SmT-infected macrophages may be one of the pathogenic mechanisms of M. aviurn.
The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymeras... more The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indica...
Background The rise in non-tuberculous mycobacteria (NTM) diseases has been reported worldwide, b... more Background The rise in non-tuberculous mycobacteria (NTM) diseases has been reported worldwide, but no major information is known for Italy. The purpose of this pilot study is to release nationwide data on distribution of NTM in our country. Methods In 2016, the Istituto Superiore di Sanità (National Institute of Health) began to collect microbiological, clinical and minimum inhibitory concentration (MIC) data on NTM infections, in collaboration with hospital laboratories located in 15 out of 20 regions (Studio Multicentrico Italiano NTM, SMI-NTM), which routinely isolate and characterize NTM by commercial identification (Genotype) and MIC (Sensititre) assays. Results In 2016-2018, 32 labs reported data on 4169 NTM strains, including 644 rapid growers (15%) and 3525 slow growers (85%). The most frequent species were Mycobacterium avium (MA) (29.2%), M. intracellulare (MI) (21.5%), M. xenopi (MX) (10.6%), M. gordonae (10.6%), M. abscessus (5.9%), M. chimaera (MC) (5.1%). Overall, 88%...
The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors ... more The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-α and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-α was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected ...
The kinetics of both intracellular and extracellular beta-lactamase production and the relationsh... more The kinetics of both intracellular and extracellular beta-lactamase production and the relationship between extracellular enzyme and in vitro susceptibility of Mycobacterium fortuitum to beta-lactam antibiotics have been studied. To this end we used a panel of stable nitrosoguanidine-induced mutants of M. fortuitum derived from the parental strain ATCC 19542 and differing in beta-lactamase production from 0.0001 to 278 U/liter in Mueller-Hinton broth. For overproducers of beta-lactamase (mutants A188, B180, C207, D316, and E31), MICs of benzylpenicillin, amoxicillin, ampicillin, and cephaloridine progressively increased with the amount of enzyme released into the medium, whereas MICs of imipenem and cefoxitin did not. The resistance of the mutants to amoxicillin was reduced up to 32-fold by clavulanic acid, whereas that to ampicillin was reduced 8-fold by sulbactam. These data suggest that the enzyme participated in the mechanisms of resistance to the beta-lactam antibiotics. Howeve...
Twenty-five strains of Mycobacterium avium-intracellulare (MAC) isolated from acquired immunodefi... more Twenty-five strains of Mycobacterium avium-intracellulare (MAC) isolated from acquired immunodeficiency syndrome (AIDS) patients in three medical centres in Italy have been studied. Serotyping performed on eighteen strains showed various serovars within either M. avium or M. intracellulare serotypes and with serovars 1 and 21 being the most prevalent (four strains for each serovar). Among fourteen drugs used for testing the antibiotic sensitivity, rifapentine, rifabutin and clofazimine showed to have the best in vitro activity. In an ex vivo model of infection using peritoneal resting macrophages from the C57BL/6 mouse, the intracellular viability of a strain of M. avium (strain 489, serovar 3) was reduced by clofazimine, amikacin, ciprofloxacin, rifabutin and clarithromycin (99, 98, 93, 89 and 69%, respectively), thus indicating for clofazimine a good correlation between in vitro and ex vivo activity.
Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs sh... more Monotherapy with isoniazid or amikacin or clarithromycin or combinations of two of these drugs showed nil to modest therapeutic activity in beige mice infected with Mycobacterium avium. However, the combination of all three, isoniazid-amikacin-clarithromycin, markedly reduced CFUs in both spleens and lungs after 91 days of infection.
The activity of clarithromycin and five other antimicrobial agents, namely amikacin, rifampicin, ... more The activity of clarithromycin and five other antimicrobial agents, namely amikacin, rifampicin, rifabutin, clofazimine and ciprofloxacin, was assessed both by an agar dilution and a radiometric method in broth on 11 Mycobacterium avium-intracellulare complex (MAC) strains, recently isolated from AIDS patients. Minimum inhibitory concentrations (MICs) radiometrically determined were, in general, several times lower than MICs assessed in agar, probably because of a partial degradation of antimicrobials during the long incubation period needed for tests in solid medium. When tested in broth, rifabutin and clofazimine showed very low MICs 90 (0.24 and 0.78 microgram/ml, respectively). Ciprofloxacin and clarithromycin also had MICs90 in the range of peak serum levels (1.93 and 3.76 micrograms/ml, respectively). Moreover, all these antimicrobials are known to concentrate several times in macrophages. MICs90 were higher for amikacin (11 micrograms/ml) and for rifampicin (8 micrograms/ml). When clarithromycin was tested against three MAC strains in combination with another drug, it showed a synergistic effect only when combined with rifampicin. Some synergistic effect was observed also when combining clarithromycin with rifampicin and amikacin, whereas in combination with rifabutin and clofazimine there was only an additive effect.
A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical propert... more A beta-lactamase from Mycobacterium fortuitum D316 was purified and some physico-chemical properties and substrate profile determined. On the basis of its N-terminal sequence and of its sensitivity to beta-iodopenicillanate inactivation, the enzyme appeared to be a class A beta-lactamase, but its substrate profile was quite unexpected, since nine cephalosporins were among the eleven best substrates. The enzyme also hydrolysed ureidopenicillins and some so-called ‘beta-lactamase-stable’ cephalosporins.
Introduzione. La borreliosi di Lyme è un'infezione multisistemica trasmessa dalla zecca (Ixodes) ... more Introduzione. La borreliosi di Lyme è un'infezione multisistemica trasmessa dalla zecca (Ixodes) e causata dalla spirocheta Borrelia burgdorferi sensu latu (BB) che provoca patologie cutanee, e, se non trattata, cardiache, reumatologiche e neurologiche. Questa infezione è endemica nell'area del triveneto. La neuroborreliosi di Lyme (LN) è molto rara e la sintomatologia può a volte essere confusa con la malattia di Parkinson (PD) o altre patologie neurodegenerative. Metodi. Da un paziente (anni 57 M) con diagnosi clinica di PD deceduto per polmonite sono stati effettuati prelievi autoptici da corteccia cerebrale, cervelletto, ponte, nervo ottico, poligono di Willis, arteria coronaria, aorta toracica e addominale e arteria renale. I prelievi sono stati divisi in 2 parti, per l'estrazione e amplificazione del DNA (nested-PCR) e per PCR in situ. È stata ricercata una sequenza conservata del gene codificante per la flagellina di BB. Come controllo è stato utilizzato un ceppo di BB ATCC. Gli amplificati sono stati sequenziati per conferma. Risultati. Tutti i campioni, ad esclusione del poligono di Willis, sono risultati positivi all'amplificazione sia in provetta che in situ. La sequenza ha confermato la provenienza del DNA da BB. Conclusioni. Il paziente ha evidenziato la presenza di una infezione sistemica da BB e quindi potrebbe presumibilmente avere sviluppato una LN con sintomatologia analoga a quella associata al PD. Da questo studio emerge l'utilità di una valutazione sierologica e molecolare per la determinazione della presenza di BB in caso di sospetto di patologia neurodegenerativa non altrimenti spiegabile.
The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and... more The groES gene of Mycobacterium avium strain 485 was cloned and expressed in Escherichia coli and the recombinant GroES protein was purified by affinity chromatography. The GroES preparation showed high purity by electrophoresis and immunoblotting. Immuno-electron microscopy showed that GroES was located both in the cytoplasm and on the surface of the mycobacterial cells and thus is readily available to interact with the host immune system. BALB/c mice were immunised intranasally with recombinant GroES, alone or in combination with a synthetic oligodeoxynucleotide containing unmethylated CpG motifs, and tested for protection against infection with M. avium. Neither GroES nor CpG alone provided any protection against subsequent challenge with M. avium, whereas a combination of the two significantly protected the lungs and spleen against colonisation by M. avium after intranasal challenge with a low dose of the organism. This indicates that intranasal administration of GroES and CpG o...
The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection rema... more The possible contribution of NKT cells to resistance to Mycobacterium tuberculosis infection remains unclear. In this paper we characterized the V␣14 NKT cell population following infection with Mycobacterium bovis bacillus Calmette-Guérin (BCG). BCG infection determined an early expansion of V␣14 NKT cells in liver, lungs, and spleen, which peaked on day 8 and was sustained until day 30. However, an NK1.1 ؉ V␣14 NKT population preferentially producing IFN-␥ predominated at an early stage (day 8), which was substituted by an NK1.1 ؊ population preferentially producing IL-4 at later stages (day 30). Despite the fact that V␣14 NKT cell-deficient mice eliminated BCG as did control mice, they had significantly higher numbers of granulomas in liver and lungs. Additionally, while control mice developed organized small granulomas, those in V␣14 NKT-deficient mice had signs of caseation, large cellular infiltrates, and some multinucleated macrophages, suggesting that V␣14 NKT cells may actually work as anti-inflammatory cells by limiting excessive lymphocyte influx and tissue pathology. In agreement, we found an increased spontaneous production and mRNA expression of TNF-␣ in liver and lungs of V␣14 NKT-deficient mice, whose neutralization in vivo by anti-TNF-␣ mAbs consistently reduced the number of granulomas in liver and lungs. Together, our results support a regulatory role for V␣14 NKT cells in the course of BCG infection through their ability to limit the extent of inflammatory response and point to an important role for this cell subset as a regulator of the balance between protective responses and immunopathology.
The therapeutic effect of 18 anti-Mycobacterium avium regimens was examined in beige mice after 9... more The therapeutic effect of 18 anti-Mycobacterium avium regimens was examined in beige mice after 91 days of infection. Treatments included monotherapy with clarithromycin (CLA), ethambutol (EMB), amikacin (AMI), rifabutin (RFB), ciprofloxacin (CIP), clofazimine (CLO), and combinations of CLA, CLA-EMB, or CLA-AMI with one of the other drugs. After monotherapy, only AMI and CLA displayed bacteriostatic and/or moderate bactericidal effects in spleens and lungs, while CIP and RFB were totally inactive and CLO and EMB showed intermediate effects against the isolate tested. Resistant mutants were isolated in spleens of mice treated with EMB, CIP, RFB, and CLO. Among two-drug combinations, CLA-RFB, CLA-CIP, and CLA-CLO were significantly more active than RFB, CIP, CLO, respectively, but not more active than CLA alone, in both organs; CLA-AMI and CLA-EMB were bactericidal in spleens and lungs, respectively. Although activity of CLA-EMB was significantly potentiated by RFB and CLO in spleens and lungs, that of CLA-AMI was significantly increased by RFB and CLO only in lungs. The most active regimen in spleens and lungs on day 91 was the combination of all three, namely CLA-AMI-EMB, which reduced the CFU numbers of 2.7 and 7.5 logio; ui comparison with day 1 and day 91 counts in untreated control mice, respectively. an antimicrobial agent known to be bactericidal in vitro and in vivo against MAC911 showed activity similar to that of CLA when tested in a beige mouse model5 of disseminated infec-tion11 and prevented the emergence of resistant mutants.11,13 In the present study, we evaluated the anti-MAC activity of 18 different regimens, including monotherapy with CLA, AMI, EMB, rifabutin (RFB), ciprofloxacin (CIP), and clofazimine (CLO) and treatment with various two-and three-drug combinations, in the beige mouse model. In particular, we compared the efficacy of three-drug combinations obtained by combining CLA-AMI and CLA-EMB with one of the remaining drugs. Finally, the emergence of drug-resistant mutants during therapy with single or combinations of drugs was investigated.
BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Myc... more BALB/c and severe combined immunodeficient (SCID) mice were inoculated intraperitoneally with Mycobacterium avium and the numbers of cfu were monitored for 70 days in spleen, liver, lung, kidney, brain and peritoneum. While BALB/c mice formed typical granulomas and controlled bacterial growth in organs, a delay in development of lesions and a modest containment of infection were observed in SCID mice. In the spleen of BALB/c mice, in which bacterial growth was contained, macrophages (Me) and natural killer (NK) cell numbers increased 24.2 times and T-and B-cell numbers increased 31.8 times after 42 days of infection; conversely, a low recruitment of mononuclear cells was observed in the spleen of SCID mice, where M. avium proliferated efficiently. Unlike visceral organs, a pronounced decrease in the number of cfu was observed in the peritoneum of BALB/c mice, concomitantly with a 331.7-fold increase in Me and NK cells and a 39.1-fold increase in T and B cells. In the peritoneum of SCID mice only a bacteriostatic effect was observed despite a 356.7-fold increase in Me and NK cells and a 222.3-fold increase in T and B cells. These results suggest that while an intact immune response can efficiently control M. avium infection in the spleen and peritoneum of BALB/c mice, cells of the innate immune system such as Me and NK cells play a role in the containment of bacterial growth in the peritoneum, but not spleen, of SCID mice.
Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 ... more Non-peptide antigens (e.g. glycolipids of microbial origin) presented by monocyte-associated CD1 molecules to T cells appear to play an important role in host immunity against tuberculosis and other pathogenic bacteria. Since vaccination with Bacillus Calmette-Guerin (BCG) has limited efficacy, the influence of viable BCG organisms on the induction of CD1b antigen by granulocyte macrophage-colony stimulating factor (GM-CSF) has been tested in adherent mononuclear cells obtained from peripheral blood of healthy donors. The results indicate that the vaccine reduces substantially CD1b induction by GM-CSF. On the other hand, BCG was found to promote a slight increase in the expression of this molecule on target cells not exposed to GM-CSF. Attempts to reverse the antagonistic effects of BCG on GM-CSF with high concentrations of GM-CSF, alone, or associated with IL-4, were unsuccessful. Moreover, mycobacteria suppression by 10 microg/ml of rifampin, did not affect BCG influence on CD1b induction. The present results suggest that mycobacterium-induced impairment of the CD1 system could play a role in the unsatisfactory results obtained with BCG vaccination.
Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy... more Clarifying how an initial protective immune response to tuberculosis may later loose its efficacy is essential to understand tuberculosis pathology and to develop novel vaccines. In mice, a primary vaccination with Ag85B-encoding plasmid DNA (DNA-85B) was protective against Mycobacterium tuberculosis (MTB) infection and associated with Ag85B-specific CD4 + T cells producing IFN-g and controlling intramacrophagic MTB growth. Surprisingly, this protection was eliminated by Ag85B protein boosting. Loss of protection was associated with a overwhelming CD4 + T cell proliferation and IFN-g production in response to Ag85B protein, despite restraint of Th1 response by CD8 + T cell-dependent mechanisms and activation of CD4 + T cell-dependent IL-10 secretion. Importantly, these Ag85B-responding CD4 + T cells lost the ability to produce IFN-g and control MTB intramacrophagic growth in coculture with MTB-infected macrophages, suggesting that the protein-dependent expansion of non-protective CD4 + T cells determined dilution or loss of the protective Ag85B-specific CD4 + induced by DNA-85B vaccination. These data emphasize the need of exerting some caution in adopting aggressive DNA-priming, protein-booster schedules for MTB vaccines. They also suggest that Ag85B protein secreted during MTB infection could be involved in the instability of protective antituberculosis immune response, and actually concur to disease progression.
It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhib... more It is widely assumed that the high level of intrinsic resistance to beta-lactam antibiotics exhibited by mycobacteria results from the combination of factors including permeability to the drugs, beta-lactamase production, and affinity for penicillin-binding proteins (PBPs). We conducted an evaluation of the second and third factors by isolating nitrosoguanidine-induced mutants from the beta-lactamase-producing strain Mycobacterium fortuitum ATCC 19542 that displayed either elevated or reduced resistance to various beta-lactam antibiotics. The mutants studied included D1 (a beta-lactamase producer with high penicillin resistance), gamma 27 (a low-level beta-lactamase producer with low penicillin resistance), and D316 (a high-level beta-lactamase producer with high penicillin resistance). In all strains examined, four major PBPs, named 1, 2a, 2b, and 3, with apparent molecular weights of 102,000, 90,000, 87,000, and 50,000, respectively, were found. The MICs of various beta-lactams to...
Both smooth transparent (SmT) and smooth domed-opaque (SmD) colonial variants were obtained from ... more Both smooth transparent (SmT) and smooth domed-opaque (SmD) colonial variants were obtained from a strain of Mycobacterium avium isolated from a patient with AIDS. The two variants showed similar biochemical characteristics but SmT bacteria proliferated better than SmD bacteria inside human macrophages and were much less capable than the SmD variant of inducing the release of IL-lp, IL-6, TNF-a, GM-CSF and G-CSF, after incubation for either 3 or 6 days. As cytokines are important extracellular signals for immune cells, the lack of induction observed in SmT-infected macrophages may be one of the pathogenic mechanisms of M. aviurn.
The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymeras... more The antituberculosis (anti-TB) drug rifampin (RIF) binds to the beta subunit of the RNA polymerase (RpoB) of Mycobacterium tuberculosis, but the bactericidal responses triggered after target interaction are not known. To evaluate whether RIF induced an oxidative burst, lysates of RIF-treated M. tuberculosis were tested for determination of reactive oxygen species (ROS) by the electron paramagnetic resonance (EPR) technique using 1-hydroxy-3-carboxy-pyrrolidine (CPH) and 5,5-dimethyl-1-pyrrolidine-N-oxide (DMPO) as spin traps. M. tuberculosis killing by RIF stimulated an increase in the rate of formation of the CPH radical (CP·). Lysate pretreatment with the O2·(-) and ·OH scavengers superoxide dismutase (SOD) and thiourea (THIO), respectively, or with the metal chelator diethylene triamine pentaacetic acid (DTPA) inhibited CP· formation, arguing in favor of a metal-catalyzed ROS response. Formation of CP· did not increase following treatment of RIF-resistant strains with RIF, indica...
Background The rise in non-tuberculous mycobacteria (NTM) diseases has been reported worldwide, b... more Background The rise in non-tuberculous mycobacteria (NTM) diseases has been reported worldwide, but no major information is known for Italy. The purpose of this pilot study is to release nationwide data on distribution of NTM in our country. Methods In 2016, the Istituto Superiore di Sanità (National Institute of Health) began to collect microbiological, clinical and minimum inhibitory concentration (MIC) data on NTM infections, in collaboration with hospital laboratories located in 15 out of 20 regions (Studio Multicentrico Italiano NTM, SMI-NTM), which routinely isolate and characterize NTM by commercial identification (Genotype) and MIC (Sensititre) assays. Results In 2016-2018, 32 labs reported data on 4169 NTM strains, including 644 rapid growers (15%) and 3525 slow growers (85%). The most frequent species were Mycobacterium avium (MA) (29.2%), M. intracellulare (MI) (21.5%), M. xenopi (MX) (10.6%), M. gordonae (10.6%), M. abscessus (5.9%), M. chimaera (MC) (5.1%). Overall, 88%...
The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors ... more The bacterial growth and the production of tumor necrosis factor alpha (TNF-α) and TNF receptors (TNF-Rs) in the spleen and blood of BALB/c mice challenged with Mycobacterium avium complex (MAC) were monitored. Infection developed in two phases: the first, up to day 21, was associated with rapid MAC multiplication in the spleen and a drop in the mycobacteremia, and the second was associated with control of the infection in both compartments. In the spleen, TNF-α and TNF-RII mRNA levels peaked on day 21 and then slowly decreased; however, no increase in the level of TNF-RI mRNA was observed throughout these experiments. The level of circulating soluble TNF-RII (sTNF-RII) was transiently increased after day 21. In a model in which overproduction of bioactive TNF-α was triggered in response to a second infection with MAC, an increased production of sTNF-RII by cultured splenocytes was also observed. Administration of an antagonist anti-TNF-RII monoclonal antibody (MAb 6G1) to infected ...
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Papers by L. Fattorini